Neonatal seizures lead to cerebrovascular disease that can produce lifelong neurological complications. Preventing cerebral vascular dysfunction may translate into prevention of neonatal encephalopathy. We search for interventions that prevent cerebral vascular injury caused by neonatal seizures. Our major focus is on cerebral vascular endothelium, which is most vulnerable to oxidative damage associated with neonatal seizures. In our previous NIH-funded studies, we have discovered the cerebroprotective role of heme oxygenase (HO) that catalyzes the formation of gaseous mediator carbon monoxide (CO) from the heme substrate. The HO/CO system provides a potent antioxidant defense mechanism in the neonatal cerebral circulation. Our exciting novel preliminary data indicate that endothelial HO activity is upregulated my mild cooling, thus producing CO elevation in cerebral microcirculation. Our preliminary findings also suggest that mild cooling reduces brain oxidative stress and prevents oxidative injury to cerebrovascular endothelium. Furthermore, we, for the first time, identified functional interaction between HO activation and the cold-sensing receptor TRPM8 expressed in cerebral vascular endothelium. These novel findings provide a strong scientific premise to our mainstream hypothesis that cooling provides endothelial vasoprotection by upregulating the HO-based antioxidant defense via a mechanism that involves activation of the cold-sensing receptor TRPM8. We will test four specific hypotheses using in vivo and in vitro approaches in newborn pigs, isolated cerebral vessels, and freshly isolated and primary cultured cerebral microvascular endothelial cells: 1) Endothelial HO is activated by mild cooling; 2) TRPM8 is critical for upregulation of endothelial HO by cooling; 3) Mild cooling promotes endothelial survival during oxidative stress via the functional link between TRPM8 and HO; and 4) Head cooling during seizures reduces oxidative stress and provides endothelial vasoprotection in the neonatal cerebral circulation. We will use an exceptional combination of complementary techniques in a clinically relevant large animal model of neonatal cerebrovascular disease. Such research is unique, as it combines functional and mechanistic studies in intact cerebral circulation with investigation of the cellular and molecular mechanisms. Pharmacological and gene silencing approaches in newborn piglets supplemented by the studies in TRPM8 KO mice will be used to pinpoint causative functional relationships among cooling, TRPM8 activation, HO activity, and endothelial functions in cerebral blood flow regulation and the blood-brain barrier integrity. The proposed project uncovers novel fundamental temperature-sensitive mechanisms of anti-oxidant cerebroprotection. This project may also have clinical implications for reducing debilitating effects of epileptic seizures in the neonatal cerebral circulation. By protecting cerebral blood flow regulation and blood-brain barrier integrity, head cooling may reduce neurological sequelae and improve the neurodevelopmental outcome of seizures.